Thermo Scientific SOLAµ SPE Plates Technical Guide. Consistent excellence. for bioanalysis

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1 Thermo Scientific SOLAµ SPE Plates Technical Guide Consistent excellence for bioanalysis

2 SOLAµ - delivering reproducible low volume extractions. Everytime! Thermo Scientific SOLAµ plates are designed for bioanalytical and clinical research analyst s who require cleaner, highly reproducible and robust sample extraction at very low sample and solvent volumes in high throughput workflows. SOLAµ achieves this due to the unique and innovative frit-less SPE technology. SOLAµ is the first micro product to truly meet the requirements of the bioanalyst. Pharmaceutical and Biopharmaceutical analytical challenges The modern bioanalytical and clinical research laboratory must provide high quality analytical results from complex biological samples in a high throughput environment while complying with strict legislation. These demands are compounded by the continued drive to higher efficacy drugs and long acting formulations which continue to push the required quantification limits to lower levels. There is also the desire to take advantage of the replacement, refinement and reduction policy. The growth of biopharmaceuticals also brings into consideration additional analytical challenges such as solvation and non-specific binding.

3 What is required of the bioanalytical method to meet these demands? Robustness low analytical failure rates Ability to process low sample volumes High sensitivity High reproducibility Ease of use High throughput processing Efficient and fast The micro SPE format is uniquely positioned to deliver on these requirements. The proprietary SOLA manufacturing process generates an SPE micro product which eliminates the issues with traditional loose-packed micro formats. By combining the support material and active media components into a solid uniform sorbent bed we remove the need for frits (Figure 1). Stable and controllable flow through the SPE micro device is another key factor controlling reproducibility of the final analytical method. This is especially important in low bed weight devices where flow control is more difficult due to lower back pressure from the sorbent. The macro-porous structure found within SOLAµ is defined by a well controlled, reproducible manufacturing process which results in uniformity well to well, plate to plate and batch to batch. This provides an added advantage when dealing with viscous biological samples, preventing blocking and enabling high throughput processing of samples. Figure 1: SOLAµ SPE design limiting issues associated with conventional SPE formats Reproducible positioning time after time No voiding No channeling Reproducible packing time after time

4 SOLAµ provides you with reproducible sensitivity Concentration of a large sample volume to achieve quantitation limits Up to 20 times increase in sensitivity can be achieved by loading a large volume of sample and eluting in a low volume. In the following example 500 µl human plasma was loaded onto the SOLAµ plate for the analysis of niflumic acid. The compound was eluted in 25 µl providing a 20 times increase in concentration whilst maintaining excellent precision. Sample preparation protocol Sample enrichment (20 x preconcentration) Relative Abundance 40 pg/ml 500 μl sample 40 pg/ml 500 μl sample pg/ml 400 pg/ml aqueous solution of niflumic acid taken through the extraction procedure (20 concentration factor) 400 pg/ml aqueous solution of niflumic acid injected neat (no concentration factor) conventional scale SPE 500 μl SOLAμ 20 pg/ml 25 μl Time (Minutes) 40 pg/ml 20 x concentration 800 pg/ml The problem; improvement in assay sensitivity required SOLAμ solution; SOLAμ for up to 20 times increase in concentration of sample without changes to workflow Peak Area counts Peak Area 7215 counts Sample pre-treatment 500 µl of human plasma diluted 1:1 with 4% phosphoric acid Sample preparation Compound(s): Matrix: Condition: Equilibrate: Load: Wash: Elute: Direct injection of eluent HPLC system: Column: Guard column: niflumic acid, niflumic acid d5 (IS) human plasma SOLAµ WAX 96 well plate ( ) 200 µl methanol 200 µl 4% phosphoric acid apply sample at 0.5 ml/min 200 µl 25 mm ammonium acetate (ph4) 200 µl 70% methanol (ph4) µl 50/50 methanol/acetonitrile with 2% ammonia Thermo Scientific Dionex UltiMate 3000 RSLC system Thermo Scientific Accucore RP-MS HPLC column 50 mm 2.1 mm 2.6 µm ( ) Thermo Scientific Accucore RP-MS Defender guard cartridge ( ) Thermo Scientific Uniguard drop-in guard holder (852-00) Mass spec system: Thermo Scientific TSQ Vantage Triple Stage Quadruple mass spec Precision Data for Niflumic Acid Peak Area Ratio (%RSD) n = 18 Recovery of Niflumic Acid (%) Matrix Effects (%) QC Low (0.4ng/mL) QC High (30ng/mL) Precision, recovery and matrix effects data for niflumic acid at Low QC 0.4ng/mL and High QC 30ng/mL (n=18)

5 Sample limited assays or scaling down a conventional SPE method and obtaining equivalent sensitivity SOLAµ allows users to directly scale down the volumes used in their analytical methods, allowing for a reduction in sample usage and eliminating issues caused by evaporation without compromising the sensitivity of their assay. This is also an important consideration when sample volumes are limited. The following example shows that by loading 25 µl of niflumic acid sample onto the SOLAµ plate and eluting in a total of 25 µl a ten-fold decrease in sample volume was achieved when compared to a traditional scale higher bed weight product. Equivalent method performance and high levels of reproducibility provided by SOLA technology were still maintained. Sample preparation protocol 250 μl sample 500 μl diluted traditional scale SPE 250 μl 250 μl ready to inject The problem; Sample volume requires reduction without compromising sensitivity Sample pre-treatment Human plasma diluted 1:1 with 4% phosphoric acid Sample preparation Compound(s): Matrix: niflumic acid, niflumic acid d5 (IS) human plasma SOLAµ WAX 96 well plate ( ) Condition: 200 µl methanol Equilibrate: 200 µl water 25 μl sample 50 μl diluted SOLAμ 25 μl 25 μl ready to inject SOLAμ solution; 10 fold reduction in sample volume with no additional step Load: Wash: Elute: Direct injection of eluent HPLC system: apply 25 µl sample at 0.5 ml/min 200 µl 25 mm ammonium acetate (ph4) 200 µl methanol µl methanol with 2% ammonia Thermo Scientific Dionex UltiMate 3000 RSLC system Equivalency of results obtained with niflumic acid (500 ng/ml) extracted with 10 mg SOLA WAX using 250 µl of sample and SOLAµ WAX using 25 µl of sample. Intensity 500, , , , , , , , ,000 50, , , , , , , , , ,000 50, Time (Minutes) SOLA SOLAµ Column: Guard column: Thermo Scientific Accucore RP-MS HPLC column 50 mm 2.1 mm 2.6 µm ( ) Thermo Scientific Accucore RP-MS Defender guard cartridge ( ) Thermo Scientific Uniguard drop-in guard holder (852-00) Mass spec system: Thermo Scientific TSQ Vantage Triple Stage Quadruple mass spec Precision Data for Niflumic Acid Analyte Peak Area (%RSD) Peak Area Ratio (%RSD) Low QC High QC Precision data niflumic acid at Low QC 0.4ng/mL and High QC 30ng/mL (n=18)

6 Extracting samples which are susceptible to solvation and non-specific binding issues With traditional SPE the eluted sample is typically blown down to increase the concentration of the sample and thus improve the sensitivity. This causes an issue for certain compound types which can be lost during this step resulting in reduced sensitivity. SOLAμ allows the sample to be extracted without the need for dry down and reconstitution. Not only does this maximize recovery of the analytes it also improves workflow efficiency and increases productivity. In the case of extraction of ibuprofen a four-fold pre-concentration was achieved without the need for dry down by loading 200 µl of sample onto the SOLAµ plate and eluting in a total of 50 µl. The results demonstrate that even with this low volume, excellent reproducibility was achieved. Sample preparation protocol Example chromatogram at quantitation limit of 10 ng/ml for ibuprofen Relative Intensity Conventional scale SPE 10 ng/ml 5 ng/ml 500 μl 10 ng/ml SOLAμ 5 ng/ml 50 μl ng/ml 40 ng/ml Time (Minutes) Re-con in 50 μl 40 ng/ml The problem; Workflow requires dry down to achieve sensitivity SOLAμ solution; Low volume allows removal of dry down Sample pre-treatment 200 µl of rat plasma diluted 1:1 with 4% phosphoric acid Sample preparation Compound(s): Matrix: Conditioning: Application: Washing: Elution: Dilution: ibuprofen, ibuprofen d3 (IS), rat plasma SOLAµ SAX 1 ml 96 well plate ( ) 200 µl methanol 200 µl water Direct injection of eluent HPLC system: Column: Guard column: load sample at 0.5 ml/min 200 µl water with 1% NH4 200 µl methanol with 1% NH μl 50/50 methanol/acetonitrile with 2% formic acid add 50 µl water to each sample Thermo Scientific Dionex Ultimate 3000 RS system Accucore RP-MS 50 mm 2.1mm 2.6 µm ( ) Accucore RP-MS defender ( ) Uniguard drop-in guard holder (852-00) Mass spec system: Thermo Scientific TSQ Vantage Triple Stage Quadruple mass spec Low QC (25 ng/ml) High QC (750 ng/ml) Ibuprofen (%RSD) n=18 Ibuprofen recovery (%) Precision data for ibuprofen at Low QC 25 ng/ml and High QC 750 ng/ml (n=18)

7 SOLAµ loading capacity The utilization of our advanced polymeric technologies in SOLAμ provides an SPE phase with excellent loading capacity. This ensures that good retention of analyte and removal of matrix interferences is achieved when a larger range of sample volumes are applied. In the following example incremental volumes of human plasma spiked at 200 ng/ml with a polar (atenolol) and non polar (imipramine) analyte were extracted. Recovery and matrix effects were monitored across the loading range to demonstrate acceptable assay performance. 120% 100% 80% 60% 40% Loading capacity: recovery and reproducibility 20% 0% 40% 30% 20% 10% 0% -10% -20% -30% -40% Undiluted plasma lead volume (µl) Matrix effects atenolol imipramine Conclusion SOLAµ meets bioanalytical needs by providing: A robust low sample volume preparation platform Reproducibility at low sample and solvent levels Processing of low volume samples Sample enrichment (20 times) Mitigates against solvation and non-specific binding issues *NB to view the complete application note please visit

8 Ordering information Description Part Number SOLAμ HRP 96 well plate SOLAμ SCX 96 well plate SOLAμ SAX 96 well plate SOLAμ WCX 96 well plate SOLAμ WAX 96 well plate Complimentary products Description Part Number HyperSep-96 Well Plate Manifold Vacuum Pump, European Version Vacuum Pump, North American Version A comprehensive product offering for your complete bioanalytical workflow Sample Preparation Sample Handling LC Columns and Accessories Separation Analysis Data Processing thermoscientific.com/sola-spe thermoscientific.com/chromatography 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. USA and Canada Australia (free call domestic) China (free call domestic) France +33 (0) Germany +49 (0) or 21 India Japan (free call domestic) fax United Kingdom +44 (0) New Zealand (free call domestic) Singapore All Other Enquiries +44 (0) Technical Support For advice and support, please visit our website: TG20993-EN 0714S